The chemical structure of PQQ (pyrroloquinoline quinone) was identified in 1979 as the third coenzyme of dehydrogenases. The presence of PQQ is confirmed mainly in dehydrogenases of many organisms, typically methanol dehydrogenase of methanol assimilating bacteria, alcohol dehydrogenase of Acetobacteria, and GDH.
Since these dehydrogenases are capable of reducing an artificial electron acceptor, they are detectable by visible light with high sensitivity, when using a dye such as nitro blue tetrazolium. Also, these dehydrogenases undergo one-way reaction, unlike AND-dependent dehydrogenases which undergo equilibrium reaction. For these reasons, these dehydrogenases are reported to be very useful for microdetermination of a compound (Methods Enzymol. vol. 89, 20 (1982)).
The most useful enzyme having PQQ as a prosthetic group is PQQ-dependent GDH, which can be utilized for determination of blood sugar. The PQQ-dependent GDH has wide applications, for example, as a dry reagent immobilized on a membrane for color reaction or as a sensor immobilized on a chip, besides the ordinary application as a biochemical reagent. As compared with glucose oxidase or NAD(P)-dependent GDH which exert the same action on glucose, the PQQ-dependent GDH is unsusceptible to dissolved oxygen, and undergoes a simple reaction which can be conducted with a simple, inexpensive device.
On the other hand, the PQQ-dependent GDH is known to have a lower stability than glucose oxidase, hexokinase or NAD(P)-dependent GDH used in glucose measurement. The mechanism of inactivation is specifically described in, for example, Biochem. J., 261,415 (1989). As to stabilization of the PQQ-dependent GDH, for instance, Arch. Biochim. Biophys., 218,623 (1982) discloses use of PQQ, and Japanese Unexamined Patent Publication No. 1997-140378 teaches combined use of calcium ions, and at least one of glutamic acid, glutamine and lysine, for instance.
However, most of the reports on stabilization of the PQQ-dependent GDH are concerned with a stabilizing method performed in a liquid, for example, a buffer solution. In view of the fact that enzymes as materials for diagnostic reagents are marketed generally in powder form (occasionally in glandular form), the reported methods are not always applicable to stabilization of enzyme products. Further, there are compounds effective for stabilization in liquid form, but liable to absorb moisture when mixed with a protein. It is often the case that such compounds rather lower the stability. In other words, there has been no report on a satisfactory method for stabilizing a PQQ-dependent GDH in the form of a powder, such as a lyophilizate.